Isolation of Bovine Foamy Virus in Japan
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FULL PAPER Virology Isolation of bovine foamy virus in Japan Yuma HACHIYA1), Kumiko KIMURA2), Keisuke OGUMA1), Mamiko ONO1), Tetsuya HORIKITA1) and Hiroshi SENTSUI1)* 1)Department of Veterinary Medicine, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, Japan 2)National Institute of Animal Health, National Agriculture and Food Research Organization (NARO) 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan ABSTRACT. Bovine foamy virus (BFV) is endemic in many countries, but has not been reported in Japan. A syncytium-forming virus was isolated from peripheral blood leukocytes of clinically J. Vet. Med. Sci. healthy cattle on a farm in Kanagawa prefecture during a periodic epidemiological survey of 80(10): 1604–1609, 2018 viral diseases. The isolate was propagated in primary fetal bovine muscle cells and subsequently passaged in Madin–Darby bovine kidney cells. Since the isolate appeared to be distinct from the doi: 10.1292/jvms.18-0121 viruses with syncytium-forming ability previously isolated in Japan, we attempted to identify it using genomic analyses and electron microscopy. A phylogenetic analysis revealed that the isolate belongs to the bovine foamy virus cluster and is highly similar to a BFV strain isolated in China. A Received: 6 March 2018 sero-epidemiological survey was performed using agar gel immunodiffusion test with the isolated Accepted: 20 August 2018 virus as the antigen, and five of the 57 cattle tested were found to be seropositive. Published online in J-STAGE: bovine foamy virus, cattle, retrovirus, spumavirus, syncytium 3 September 2018 KEY WORDS: Foamy viruses are members of the subfamily Spumaretrovirinae within the family Retroviridae characterized by unique features of their replication strategy and molecular biology [18]. They are commonly found in primates, cats, horses, and cattle [6, 10, 24, 28, 30]. Bovine foamy virus (BFV) was first isolated from clinically normal cattle in 1969 and is present in a high percentage of livestock cattle in various parts of the world [1–3, 13–15, 21, 32]. It was initially presumed that BFV might be related to bovine lymphosarcoma, but the results of additional studies have not supported this hypothesis and the pathogenicity of BFV is unclear [4, 22]. Although foamy viruses are considered nonpathogenic, infection by these viruses may be associated with transient health abnormalities resulting from persistent infection and integration of viral DNA into the host genome [7, 19]. To date, BFV has not been isolated in Japan. Recently, we conducted an epidemiological survey on a farm in Kanagawa prefecture in Japan to identify cattle persistently infected with bovine viral diarrhea virus. A syncytium-forming virus was successfully isolated from peripheral blood leukocytes (PBLs) of clinically healthy cattle. Because the isolate appeared to be distinct from other syncytium-forming viruses previously isolated in Japan (e.g., bovine respiratory syncytial virus, bovine parainfluenza virus 3, bovine herpesvirus 1, bovine leukemia virus and bovine immunodeficiency virus), we attempted to identify the isolate and confirmed the first isolation of BFV in Japan. MATERIALS AND METHODS Blood samples and virus isolation Fifty-seven cattle, 6 sheep, and 12 goats reared on a farm in the Kanto district of Japan were used in this study. Blood was collected from the tail vein of the cattle and from the jugular vein of the sheep and goats. Plain tubes were used for serum collection and EDTA-containing tubes were used to obtain PBLs. After lysis of erythrocytes by mixing with two volumes of 0.83% NH4Cl solution containing 0.01% EDTA, PBLs were separated from blood and washed three times in PBS with centrifugation at 1,000 rpm for 10 min. PBL concentration was adjusted to 1 × 107/ml and used for polymerase chain reaction (PCR) and virus isolation. One ml of PBL suspension (1 × 107 cells) was co-cultured with 1 ml of fetal bovine muscle (FBM) cells (1.5 × 105 cells) in 6-well plates for virus isolation. After 48 hr, the infected cells were washed with Eagle’s minimal essential medium (Eagle’s MEM) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and fresh medium was added. The cells were cultured for a minimum of seven days. If a cytopathic effect (CPE) was not observed, blind passage was carried out using two methods: (i) subculture of infected cells by trypsinization, and (ii) inoculating uninfected cells with the culture medium. The passaging was performed twice. Cell culture FBM cell cultures were prepared by standard tissue-culture methods [25], and used within 20 passages for virus isolation and *Correspondence to: Sentsui, H.: [email protected] ©2018 The Japanese Society of Veterinary Science This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/) 1604 ISOLATION OF BOVINE FOAMY VIRUS viral antigen preparation. Madin–Darby bovine kidney (MDBK) cells were employed for viral antigen preparation of the isolated virus for agarose gel immunodiffusion (AGID) tests. FBM cells were cultured at 37°C in Eagle’s MEM containing 10% fetal calf serum (FCS), and MDBK cells were cultured under similar conditions in Eagle’s MEM containing 5% FCS and 0.3% tryptose phosphate broth. To observe syncytium formation, the cells were fixed with methanol when CPE was observed and stained with Giemsa. Uninfected FBM cells were used as a negative control. Serological assays A total of 57 serum samples collected from farm-reared cattle were initially used to conduct the seroprevalence survey against the BFV isolate, and subsequently, sera from sheep and goats were included as well. BFV-infected MDBK cells were used as antigens for the AGID tests. Infected cells were detached from the culture bottle using a rubber policeman when CPE had appeared in about 75% of cells, collected in a centrifugation bottle, and washed three times in PBS with centrifugation at 2,500 rpm for 10 min. Cells were resuspended in a small volume of PBS containing 0.1% triton X-100 (approximately 1/100 volume of original cell suspension culture fluid), sonicated, and used as the antigen for AGID tests. AGID tests were performed according to the methods described by Malmquist et al. [22] and Kono et al. for bovine leukemia virus [17] with minor modifications. The wells were 5 mm in diameter, and six circumferential wells were placed at a distance of 3 mm from the central well. The central well was filled with the antigen and two side wells were filled with positive control serum. The other four wells were filled with undiluted test serum samples. The gel diffusion plate was incubated at room temperature for 2 days and precipitation lines were observed. Samples were regarded as positive when a precipitation line was formed and continuously joined with the positive control line formed between the antigen well and the positive control serum well. If a precipitation line was not formed but the control line curved marginally toward the inside of the test serum well, the sample was regarded as weakly positive. A positive control antiserum for the AGID assay was identified among serum samples collected from cattle on the farm. It yielded a single dense precipitation line that was specific for the BFV isolate, as demonstrated by an identical AGID reaction observed using a post serum from a rabbit 30 days after experimental infection with the virus. Positive control sera Because rabbits are sensitive to a simian foamy virus [12], we first attempted to prepare positive control sera by infecting a rabbit. RK13 cells, originating from rabbit kidney, were inoculated with the isolated virus and using this for immunization. A rabbit was intramuscularly inoculated with 1 × 107 virus-infected RK13 cells three times at 10-day intervals. Serum taken 10 days after the final immunization formed a precipitation line in the AGID test, but the line was not clear enough to be used as a positive control. Therefore, a positive control antiserum for the AGID tests was selected among serum samples collected from cattle on the farm, tested to ensure specificity for BFV. The bovine serum formed a single dense precipitation line that connected with the line formed with the post serum of the immunized rabbit. Transmission electron microscopy For electron microscopy, infected FBM cells were detached from the petri dish using a rubber policeman. The cells were fixed with 2.5% glutaraldehyde in PBS, pelleted by centrifugation, post fixed with osmium tetroxide (1% in 0.1 M phosphate buffer, pH 7.4), dehydrated in graded ethanol, and embedded in LUVEAK-812 resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed using a transmission electron microscope (H-7500, Hitachi High-Technologies Corp., Tokyo, Japan). DNA extraction from infected cells and PBLs Infected cells were trypsinized and washed twice in PBS. Approximately 2 × 105 cells that were resuspended in PBS were transferred to 1.5 ml microcentrifuge tubes, and centrifuged at 2,000 × g for 5 min. DNA was extracted from the cell pellet using DNeasy® Blood & Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. Subsequently, PBLs were collected from cow no. 43, which induced CPE on FBM cells by the procedure described in “Blood samples and virus isolation” above, and DNA was extracted directly from approximately 1 × 106 cells and subjected to PCR. Polymerase chain reaction To amplify a region of the BFV env gene using nested PCR, we modified the method described by Materniak et al. [23]. PCR was performed using the GoTaq Green Master Mix (Promega, Madison, WI, U.S.A.). The sequences of the first PCR primers were P1 (5′-tggactctagtagtctcacc-3′) and P2 (5′-cttagaaagcgtggtaatggc-3′), and a 1,248-bp product was amplified.